Methods and reagents for the rapid and efficient isolation of circulating cancer cells
First Claim
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1. A method for detecting and enumerating rare cells in a mixed cell population, the presence of said rare cells in said population being indicative of a disease state, comprising:
- a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said rare cells;
b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with the rare cells, to the substantial exclusion of other sample components;
c) contacting said immunomagnetic sample with at least one biospecific reagent which labels said rare cells; and
d) analyzing said labeled rare cells to determine the presence and number of any rare cells in said immunomagnetic sample, the greater the number of rare cells present in said sample the greater the severity of said disease state.
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Abstract
A highly sensitive assay is disclosed which combines immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis to detect, enumerate and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood and has a greater sensitivity than conventional PCR or immunohistochemistry by 1-2 orders of magnitude. In addition, the assay facilitates the biological characterization and staging of carcinoma cells.
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Citations
83 Claims
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1. A method for detecting and enumerating rare cells in a mixed cell population, the presence of said rare cells in said population being indicative of a disease state, comprising:
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a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said rare cells;
b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with the rare cells, to the substantial exclusion of other sample components;
c) contacting said immunomagnetic sample with at least one biospecific reagent which labels said rare cells; and
d) analyzing said labeled rare cells to determine the presence and number of any rare cells in said immunomagnetic sample, the greater the number of rare cells present in said sample the greater the severity of said disease state. - View Dependent Claims (2, 4, 5, 6)
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3. A method as claimed in claim 3, wherein the volume of said immunomagnetic sample containing said enriched rare cells is reduced.
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7. A method for detecting and enumerating cancer cells in a mixed cell population, comprising:
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a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing said cancer cells;
b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with the cancer cells, to the substantial exclusion of other sample components;
c) contacting said immunomagnetic sample with at least one biospecific reagent which labels said cancer cells; and
d) analyzing said labeled cancer cells to determine the presence and number of any cancer cells in said immunomagnetic sample, the greater the number of cancer cells present in said sample the greater the severity of said cancer. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 82)
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22. A method of diagnosing early stage cancer in a test subject, comprising:
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a) obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing cancer cells;
b) preparing a cell fraction from said biological specimen, said cell fraction being enriched for cancer cells, if present in said biological specimen; and
c) analyzing said enriched fraction for the presence of said cancer cells, the presence of said cancer cells in said specimen indicating the presence of early stage cancer in said test subject. - View Dependent Claims (23, 24, 25, 26, 27)
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- 28. A coated, magnetic particle comprising a nanoparticle core of magnetic material, and a base coating material on said magnetic core in an amount sufficient to hinder non-specific binding of biological macromolecules to said magnetic core.
- 29. A coated, magnetic particle comprising a nanoparticle core of magnetic material, a base coating material that forms a discontinous coating on said magnetic core, providing at least one area of discontinuity which, if accessible, contributes to non-specific binding of said base coated particle to biological macromolecules, and an additional coating material that hinders access to said at least one area of discontinuity by said biological macromolecules.
- 44. A coated magnetic particle comprising a nanoparticle core material of a magnetic transition metal oxide, a protein base coating material and an additional coating material coupled to said base coating material through a biofunctional linking compound, said additional coating material being one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin.
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48. A test kit for screening a patient sample for the presence of circulating rare cells comprising:
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a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said rare cell, said antibody being coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a second characteristic determinant of said rare cell; and
c) a cell specific dye for excluding sample components other than said rare cells from analysis. - View Dependent Claims (49, 50)
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51. A kit for screening a patient sample for the presence of circulating tumor cells, comprising:
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a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a cancer cell determinant; and
c) cell specific dye for excluding sample components other than said tumor cells from analysis. - View Dependent Claims (52, 53, 54, 55, 56, 57)
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58. A kit for screening a patient sample for the presence of circulating breast cancer cells, comprising:
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a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a breast cancer cell determinant; and
c) cell specific dye for excluding sample components other than said breast cancer cells from analysis.
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59. A kit for screening a patient sample for the presence of circulating prostate cancer cells, comprising:
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a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a prostate cancer cell determinant; and
c) cell specific dye for excluding sample components other than said prostate cancer cells from analysis.
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60. A kit for screening a patient sample for the presence of circulating colon cancer cells, comprising:
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a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a colon cancer cell determinant; and
c) cell specific dye for excluding sample components other than said colon cancer cells from analysis.
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61. A kit for screening a patient sample for the presence of circulating bladder cancer cells, comprising:
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a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) at least one antibody having binding specificity for a bladder cancer cell determinant; and
c) cell specific dye for excluding sample components other than said bladder cancer cells from analysis.
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62. A test kit for monitoring a patient for the recurrence of cancer, said test kit comprising:
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a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
b) an antibody having binding specificity for an intracellular cytokeratin marker;
c) an antibody having binding specificity for a predetermined cancer specific determinant which is different from said intracellular cytokeratin marker; and
d) a cell specific dye for excluding sample components other than said cancer cells from analysis. - View Dependent Claims (63, 64, 65, 66, 67)
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68. A method for determining likelihood of cancer recurrence in a human subject previously treated for cancer, comprising:
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a) obtaining a blood sample from said subject;
b) determining the number of epithelial cells in said sample;
c) comparing the number of epithelial cells with a statistically determined number of epithelial cells from a group of tumor-free patient controls; and
d) assigning a likelihood of cancer recurrence when the number of epithelial cells exceeds a pre-determined value based on statistical averages of circulating epithelial cells from healthy subjects compared with statistical averages of circulating epithelial cells from cancer patients. - View Dependent Claims (69, 70, 71)
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72. A method of distinguishing an organ confined carcinoma from a carcinoma with metastatic properties, comprising:
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a) obtaining a blood sample from a test subject;
b) determining the number of circulating epithelial cells in said sample;
c) identifying the tissue origin of the epithelial cells;
d) comparing the number of circulating tissue specific epithelial cells determined for said test subject with a statistically determined average number of circulating tissue specific epithelial cells from a group of healthy subjects, a group of organ-confined carcinoma patients and a group of patients with metastatic carcinoma; and
e) identifying said human test subject cells as metastatic when the number is within the statistically determined average for the group of patients with metastatic carcinoma. - View Dependent Claims (73, 74, 75, 76)
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77. A method of following remission status in a human cancer patient undergoing cancer therapy treatment, comprising:
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a) obtaining a blood sample from a human cancer patient;
b) determining the number of circulating epithelial cells in said sample prior to and periodically subsequent to said cancer therapy treatment wherein an increase over time in the number of epithelial cells following treatment compared to the number of epithelial cells just after treatment is indicative of relapse and wherein maintenance of epithelial cell levels comparable to levels just after treatment is indicative of continuing remission. - View Dependent Claims (78)
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79. A fraction of peripheral blood enriched for circulating neoplastic cells, said enrichment being at least 2,500 fold with reference to the sample of peripheral blood from which said fraction was obtained.
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80. A fraction of peripheral blood enriched for circulating neoplastic cells, said enrichment being at least 5,000 fold with reference to the sample of peripheral blood from which said fraction was obtained.
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81. A fraction of peripheral blood enriched for circulating neoplastic cells, said enrichment being at least 10,000 fold with reference to the sample of peripheral blood from which said fraction was obtained.
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83. A method for increasing numbers of circulating epithelial cells in a blood sample, comprising massaging a tissue suspected of being cancerous prior to obtaining said blood sample.
Specification